The present invention is generally in the field of methods for making 2-hydroxyacid monomers, and the resulting polyhydroxyalkanoate polymers.
Numerous microorganisms have the ability to accumulate intracellular reserves of poly [(R)-3-hydroxyalkanoates] (“PHA”) polymers. PHAs are biodegradable thermoplastic materials, produced from renewable resources, with a broad range of industrial and biomedical applications (Williams and Peoples, 1996, CHEMTECH 26, 38-44). Around 100 different monomers have been incorporated into PHA polymers, as reported in the literature (Steinbüchel and Valentin, 1995, FEMS Microbiol. Lett. 128; 219-228) and the biology and genetics of their metabolism has recently been reviewed (Huisman and Madison, 1999, Microbiology and Molecular Biology Reviews, 63: 21-53).
Fermentation and recovery processes for a range of PHA types have been developed using a variety of bacteria including Azotobacter, Alcaligenes latus, Comamonas testosteroni and genetically engineered E. coli and Klebsiella, as recently reviewed by Braunegg et al., 1998, Journal of Biotechnology 65: 127-161; Choi and Lee, 1999, Appl. Microbiol. Biotechnol. 51: 13-21. More traditional polymer synthesis approaches have also been examined, including direct condensation and ring-opening polymerization of the corresponding lactones (Jesudason and Marchessault, 1994, Macromolecules 27: 2595-2602).
Synthesis of PHA polymers containing the monomer 4-hydroxybutyrate (PHB4HB, Doi, Y.1995, Macromol. Symp. 98, 585-599) or 4-hydroxyvalerate and 4-hydroxyhexanoate containing PHA polyesters have been described (Valentin et al., 1992, Appl. Microbiol. Biotechnol. 36, 507-514 and Valentin et al., 1994, Appl. Microbiol. Biotechnol. 40, 710-716). The PHB4HB copolymers can be produced with a variety of monomer compositions which provides a range of polymer properties (Saito, Y, Nakamura, S., Hiramitsu, M. and Doi, Y., 1996, Polym. Int. 39: 169). The homopolymer poly(4-hydroxybutyrate), or P4HB, has been synthesized in recombinant E. coli (Hein et al., 1997, FEMS Microbiol. Lett. 153:411-418) by using plasmid-borne Ralstonia eutropha PHA synthase (phaC) and the Clostridium kluyveri 4HB-CoA transferase (orfZ) genes.
PHA copolymers of 3-hydroxybutyrate-co-3-hydroxypropionate have also been described (Shimamura et. al., 1994, Macromolecules 27: 4429-4435; Cao et. al., 1997, Macromol. Chem. Phys. 198: 3539-3557). The highest level of 3-hydroxypropionate incorporated into these copolymers is 88 mol % (Shimamura et. al., 1994, Macromolecules 27: 4429-4435).
WO 02/08428A2 to Metabolix Inc. describes genetically engineered bacteria for the production of PHA copolymers from polyol feedstocks. Although over 100 different monomers have been incorporated into PHAs in organisms, the presence of glycolic acid in a biosynthetic PHA has never been reported. Glycolic acid is the simplest of the hydroxyacids, and polymers containing glycolic acid previously have been chemically synthesized. For instance, high molecular mass glycolic acid polymers are preferentially prepared by ring opening polymerization from the cyclic dimer, glycolide. Glycolic acid containing polymers are used in absorbable sutures, internal fixations devices, tissue engineering scaffolds, drug release matrices, etc. See, for example, U.S. Pat. No. 3,867,190; U.S. Pat. No. 3,736,646; Fukuzaki, et al., “A new biodegradable copolymer of glycolic acid and lactones with relatively low-molecular weight prepared by direct copolycondensation in the absence of catalysts” in J. Biomed. Mater. Res. 25(3):315-28 (1991). Synthetic polymers containing glycolic acid have been used for absorbable medical devices since the 1970's. See, for example, Chujo, et al., “Ring-opening polymerization of glycolide” in Makromol. Chem. 100:262-6 (1967); Fukuzaki, et al., “Direct copolymerization of glycolic acid with lactones in the absence of catalysts” in Eur. Polym. J. 26(4):457-61 (1990); Kricheldorf, et al., “Polylactones, 2. Copolymerization of glycolide with β-propiolactone, γ-butyrolactone or δ-valerolactone” in Makromol. Chem. 186(5):955-76 (1985); Kricheldorf, et al., “Polylactones, 3. Copolymerization of glycolide with L,L-lactide and other lactones” in Makromol. Chem. 12(Polym. Specific Prop.):955-76 (1985); Nakayama, et al., “Synthesis and biodegradability of novel copolyesters containing γ-butyrolactone units” in Polymer 39(5): 1213-1222 (1998); Nakayama, et al., “Syntheses of biodegradable polyesters and effect of chemical structure on biodegradation” in Nippon Kagaku Kaishi 1:1-10 (2001). However, copolymers formed of glycolic acid and various lactones by chemical synthesis have relatively low molecular weights. Moreover, it is difficult to control the stereo configuration of the lactone units in the copolymers formed by chemical synthesis.
It would be useful to be able to incorporate glycolic acid into PHA polymers in bacterial production systems. This would further expand the range of physical properties available from the PHA family of polymers and the presence of the glycolic acid monomers would provide a means for controlling the degradation rate of the PHAs for industrial and biomedical uses. In particular, the incorporation of glycolic acid into PHAs would provide a means of controlling the in vivo degradation rate of PHA biomedical devices using a monomer already having a history of safe use in medical implants.
It is therefore an object of the present invention to provide a method for the biosynthesis of PHAs containing glycolic acid.
It is another object of this invention to provide methods for the biosynthesis of PHAs containing glycolic acid and at least one other monomer such as 3-hydroxybutyric acid, 3-hydroxypropionic acid, 3-hydroxyvaleric acid, 3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid, 3-hydroxydecanoic acid, 4-hydroxybutyric acid or 4-hydroxyvaleric acid. Specific glycolic acid containing PHAs of interest include poly-3-hydroxybutyric acid-co-glycolic acid and poly-glycolic acid-co-4-hydroxybutyric acid.
It is further an object of the present invention to provide PHAs containing 2-hydroxyacids by biosynthesis for medical and industrial applications.